The Desmoglein 2 interactome in primary neonatal cardiomyocytes

Figures

• 

  Figure 1.. Desmosome organization and dynamics at neonatal cardiomyocyte cell-cell contacts. (A) Cartoon schematic of the cardiac desmosome showing Desmocollin 2 (DSC2), Desmoglein 2 (DSG2), Plakophilin 2 (PKP2), Plakoglobin (JUP), Desmoplakin (DSP), and Desmin (DES). (B) Thin section electron microscopy image of neonatal cardiomyocyte cell-cell junction. The cell-cell contact is highlighted in purple. Desmosomes (DE), adherens junctions (AJ), and gap junction (GJ) labeled. Scale bar is 1 micron. (C) Top, cartoon schematic of exon organization and splicing at the 3’ end of desmocollin 2 in mouse chromosome 18. Splicing of exons 15 and 17 produces variant Dsc2a with a longer C-terminal cytoplasmic tail. In contrast, splicing of exons 15, 16, and 17 introduces a new stop codon to create variant Dsc2b with a truncated C-terminal tail. PCR primer pair locations for set 1 and set 2 are shown as arrows. Bottom, PCR results from cDNA isolated from cultured neonatal cardiomyocytes and adult mouse heart. Variant Dsc2b is the primary transcript in cultured cardiomyocytes and the adult heart. (D) Images from FRAP experiments showing pre-bleach, post-bleach, and recovery after 10 minutes in…

• 

  Figure 2.. DSG2–BioID2 localizes to cell-cell contacts and labels junctional proteins. (A) Illustration of DS2G-BioID. The BioID2 biotin ligase with a C-terminal HA tag was fused to the end of the DSG2 C-terminal tail. (B) Neonatal cardiomyocytes infected with DSG2-BioID adenovirus and stained for CDH2 (green in merge) and labeled with streptavidin conjugated to Cy3 (magenta in merge) to identify biotinylated proteins. White asterisks mark infected cells. Scale bar is 10 microns. (C) Streptavidin western blot of pulldowns from control (uninfected), CDH2-BioID2 infected, or DSG2-BioID2 infected cardiomyocytes. Streptavidin-precipitated material was separated by SDS-PAGE and blotted for hemagglutinin (HA, left blot) or streptavidin (SA, right blot).

• 

  Figure 3.. Quantitative mass spectrometry identifies the DSG2 interactome. (A) Plot of p-value (−log10) versus fold-change (log2) of identified proteins. Dashed lines mark p=0.001 (y-axis) and fold-change of 10 (x-axis). (B) Summary of identified peptide and protein number at each stage of condition stringency. (C) Rank plot of abundance (iBAQ mass, log2). Proteins of interest are marked with colored circles and labeled. Circle color corresponds to the primary adhesion complex: green (desmosome), orange (adherens junction), and purple (gap junction). (D) Protein distribution by assigned category based on number (top pie chart) or abundance (iBAQ) (bottom pie chart). (E) IPA enrichment analysis of DSG2 hits in canonical signaling pathways.

• 

  Figure 4.. DSG2 and CDH2 interactome comparison. (A) Venn diagram of cardiomyocyte DSG2 interactome (325 hits, green) versus cardiomyocyte CDH2 interactome (353 hits, orange). 172 hits are shared between the two interactomes (tan). Note that the original CDH2 interactome listed 354 genes, not 353. PALM2 and PALM2-AKAP were initially designated as separate genes, but they are classified as one gene (PAKAP) in mouse. (B) Scatter plot of shared hits (black dots) based on abundance (iBAQ mass, log2) in DSG2 (x-axis) and CDH2 (y-axis) interactomes. Core desmosome and adherens junction proteins are marked in red. Linear regression analysis calculated the slope (orange line), 95% confidence intervals (dashed orange lines flanking the slope), and R2 value. Slope deviation from zero was analyzed for significance (p-value). (C) IPA enrichment analysis of DSG2 only hits (left), DSG2-CDH2 shared hits (middle), and CDH2 only hits (right) in canonical signaling pathways.

• 

  Figure 5.. Desmosome interactome comparison. (A) Venn diagram of cardiomyocyte DSG2 interactome (325 hits), DSP interactome in keratinocytes (682 hits), and the combined JUP, PKP2, and DSC2a Ca2+-dependent interactome in MDCK cells (labeled DM for desmosome; 194 hits in total). Sixteen proteins are shared between the interactomes. (B) IPA enrichment analysis of DSG2-DSP shared hits (top left), DSG2-DM shared hits (top right), DSG2-DSP-DM shared hits (bottom right), and DSG2 unique hits (bottom right) in canonical signaling pathways.

• 

  Figure 6.. Cardiomyocyte DSG2 interactome network. (A) DSG2 interactome interaction network organized into four tiers based on IPA. Published experimental data support all protein-protein interactions. Hierarchical classification was done manually around DSG2. Primary interactors bind DSG2 directly. Secondary interactors bind to primary interactors, but not to DSG2. Tertiary interactors bind secondary interactors, and quaternary interactors bind tertiary interactors. Green circles signify hits unique to the DSG2 interactome; yellow circles mark hits shared with the CDH2 interactome. The 30 most abundant DSG2 interactome proteins are outlined in orange. 39 proteins not connected to the network are listed in Table S1. (B) Percent distribution of DSG2 unique (green bar) and DSG2/CDH2 shared (yellow bar) hits within each tier.

• 

  Figure 7.. PKP2 is recruited to cardiomyocyte AJs under tension. (A-F) Cardiomyocytes were cultured for 72–96 hours, fixed, and stained for PKP2, F-actin, and CTNNB1 (A), CTNNA1 (B), CTNNA3 (C), JUP (D), AFDN (E), or GJA1 (F). All images are maximum projections of 1 micron z-stacks. Merge images show overlap between PKP2 (green) and adhesion protein (magenta) channels. (G) Pearson’s coefficient was calculated between PKP2 and individual adhesion protein channels at cell-cell contacts. Scatter plot of all data points shown. The thick horizontal blue line marks the median, and the blue error bars define the interquartile range. Compact letter display defines statistically indistinguishable coefficients (one-way ANOVA with Tukey’s multiple comparisons). (H-J) Cardiomyocytes were cultured for 72 hours, incubated for 1 hour in DMSO (control, H) or 100 μM blebbistatin (I and J), fixed, and stained for JUP, PKP2, and F-actin. (K). PKP2 average fluorescence signal intensity at cell-cell contacts divided by the average intensity in the cytoplasm (PKP contact/cytoplasm ratio) in cells treated with DMSO or blebbistatin. A scatter plot of all data points is shown, and the median and interquartile range…